The long-term goal of this proposed program is to understand the functions of Ser/Thr protein kinases (STPKs) recently discovered in Mycobacterium tuberculosis (Mtb). This pathogenic bacterium, which is the causative agent of tuberculosis (TB), infects one third of the world's population. TB kills over 3 million people annually, more than any other infectious disease. Despite the large amount of work on protein kinases and phosphatases, little is known about the roles of the Mtb STPKs and the single Mtb Ser/Thr phosphatase, Ppp. Research to define the specific biological functions of these enzymes is needed to know how environmental signals regulate Mtb metabolism. We will focus biochemical, biophysical, structural and genetic methods principally on four Mtb proteins. This research has four specific aims: 1. Define the basis for signaling in two Mtb transmembrane receptor kinases. 2. Define the mechanism of PknB kinase switching between off and on states. 3. Discover protein substrates of Mtb STPKs. 4. Define the intracellular signaling pathways, focusing on the single Mtb Ser/Thr phosphatase, Ppp. Our preliminary studies established the feasibility of these aims. Our biochemical and structural work on the kinase domain of Mtb PknB showed that the prokaryotic and eukaryotic STPKs share a remarkably conserved three-dimensional structure and universal mechanisms of regulation and substrate recognition. We determined the first crystal structures of a bacterial STPK sensor domain and bacterial Ser/Thr phosphatase. We have set the stage to define the active and inactive states of the PknB kinase. Our proposed exploration of the substrates of PknB and Ppp will begin to define the functions of these proteins. Because of the central roles of kinase signaling in cellular physiology, the increasing focus on STPKs as pharmaceutical targets, and the worldwide health impact of TB, our proposed studies will have high significance for molecular biology and medicine.